Isotopologues of 2-(tert butylamino)-4-((1R,3R,4R)-3-hydroxy-4-methylcyclohexylamino)-pyrimidine-5-carboxamide

ABSTRACT

Provided herein are isotopologues of Compound A, which are enriched with isotopes such as, for example, deuterium. Pharmaceutical compositions comprising the isotope-enriched compounds, and methods of using such compounds are also provided.

This application is a continuation of U.S. application Ser. No.15/546,885, filed Jul. 27, 2017, which is a U.S. national stageapplication of International Patent Application No. PCT/US2016/015276,filed Jan. 28, 2016, which claims the benefit of U.S. ProvisionalApplication No. 62/109,096, filed Jan. 29, 2015, the entire content ofwhich is incorporated herein by reference.

1 FIELD

Provided herein are isotopologues of certain heterocyclic carboxamides,compositions comprising the isotopologues, methods of making theisotopologues, and methods of their use for treatment or prevention ofdiseases and conditions including, but not limited to, inflammatorydiseases, autoimmune diseases, and cancers.

2 BACKGROUND

The connection between abnormal protein phosphorylation and the cause orconsequence of diseases has been known for over 20 years. Accordingly,protein kinases have become a very important group of drug targets. (SeeCohen, Nature, 1:309-315 (2002), Gaestel et al. Curr. Med. Chem. 14:2214-223 (2007); Grimminger et al. Nat. Rev. Drug Disc. 9(12):956-970(2010)). Various protein kinase inhibitors have been used clinically inthe treatment of a wide variety of diseases, such as cancer and chronicinflammatory diseases, including rheumatoid arthritis and psoriasis.(See Cohen, Eur. J. Biochem., 268:5001-5010 (2001); Protein KinaseInhibitors for the Treatment of Disease: The Promise and the Problems,Handbook of Experimental Pharmacology, Springer Berlin Heidelberg, 167(2005)).

JNK is a ubiquitously expressed serine/threonine kinase belonging,together with ERK (extracellular-regulated kinase) and p38, to thefamily of mitogen-activated protein kinases (MAPKs). (Kyriakis J M, Sci.STKE (48):pe1 (2000); Whitmarsh A J, et al. Sci. STKE (1):pe1 (1999);Schramek H, News Physiol. Sci. 17:62-7 (2002); Ichijo H, Oncogene18(45):6087-93 (1999)). MAPKs are important mediators of signaltransduction from the cell surface to the nucleus, using phosphorylationcascades to generate a coordinated response by a cell to an externalstimulus by phosphorylation of selected intracellular proteins,including transcription factors. Additionally, JNK also phosphorylatesnon-nuclear proteins, for example, IRS-1, and Bcl-2 family members.(Davis R J, Trends Biochem. Sci. 9(11):470-473 (1994); Seger R et al.,FASEB J.; 9(9):726-35 (1995); Fanger G R et al., Curr. Opin. Genet.Dev.; 7(1):67-74 (1997)).

The elucidation of the intricacy of protein kinase pathways and thecomplexity of the relationship and interaction among and between thevarious protein kinases and kinase pathways highlights the importance ofdeveloping pharmaceutical agents capable of acting as protein kinasemodulators, regulators or inhibitors that have beneficial activity onmultiple kinases or multiple kinase pathways.

The compound chemically named2-(tert-butylamino)-4-((1R,3R,4R)-3-hydroxy-4-methylcyclohexylamino)-pyrimidine-5-carboxamide(alternatively named2-[(1,1-dimethylethyl)amino]-4-[[(1R,3R,4R)-3-hydroxy-4-methylcyclohexyl]amino]-5-pyrimidinecarboxamide)and tautomers thereof are disclosed in U.S. Patent ApplicationPublication No. 2013/0029987, published on Jan. 31, 2013, andInternational Pub. No. WO2012/145569, the entireties of each of whichare incorporated by reference herein.

Citation or identification of any reference in Section 2 of thisapplication is not to be construed as an admission that the reference isprior art to the present application.

3 SUMMARY

Embodiments provided herein encompass isotopologues of Compound 1:

and pharmaceutically acceptable salts, stereoisomers, tautomers, solidforms, polymorphs, hydrates, clathrates, and solvates thereof(collectively with Compound 1 referred to herein as “Compound A”). Inone embodiment, Compound 1 is2-(tert-butylamino)-4-((1R,3R,4R)-3-hydroxy-4-methylcyclohexylamino)-pyrimidine-5-carboxamide(alternatively named2-[(1,1-dimethylethyl)amino]-4-[[(1R,3R,4R)-3-hydroxy-4-methylcyclohexyl]amino]-5-pyrimidinecarboxamide).

In certain embodiments, the isotopologue is an isotopologe of thefollowing structure:

Certain embodiments encompass mixtures of an isotopologue of Compound A.Certain embodiments encompass methods of synthesizing, isolating, orcharacterizing an isotopologue of Compound A. In certain embodiments,the isotopologues of Compound A are deuterium, carbon-13, nitrogen-15,or oxygen-18 enriched, or combinations thereof.

Further provided herein are methods for using the pharmaceuticalcompositions and dosage forms of an isotopologue of Compound A fortreating or preventing diseases or disorders treatable or preventable byinhibition of a JNK pathway, as described herein. In some embodiments,the diseases or disorders include, but are not limited to, interstitialpulmonary fibrosis, systemic sclerosis, scleroderma, chronic allograftnephropathy, antibody mediated rejection, or lupus. In otherembodiments, the diseases or disorders include, but are not limited to,liver fibrotic disorders, or diabetes and/or metabolic syndrome leadingto liver fibrotic disorders, as described herein.

The present embodiments can be understood more fully by reference to thedetailed description and examples, which are intended to exemplifynon-limiting embodiments.

4 DETAILED DESCRIPTION

The descriptions of the terminology provided below apply to the terms asused herein, unless otherwise specified.

The term “isotopic composition” refers to the amount of each isotopepresent for a given atom, and “natural isotopic composition” refers tothe naturally occurring isotopic composition or abundance for a givenatom. Atoms containing their natural isotopic composition may also bereferred to herein as “non-enriched” atoms. Unless otherwise designated,the atoms of the compounds recited herein are meant to represent anystable isotope of that atom. For example, unless otherwise stated, whena position is designated specifically as “H” or “hydrogen,” the positionis understood to have hydrogen at its natural isotopic composition.

The term “isotopically enriched” refers to an atom having an isotopiccomposition other than the natural isotopic composition of that atom.“Isotopically enriched” may also refer to a compound containing at leastone atom having an isotopic composition other than the natural isotopiccomposition of that atom. As used herein, an “isotopologue” is anisotopically enriched compound.

The term “isotopic enrichment” refers to the percentage of incorporationof an amount of a specific isotope at a given atom in a molecule in theplace of that atom's natural isotopic composition. For example,deuterium enrichment of 1% at a given position means that 1% of themolecules in a given sample contain deuterium at the specified position.Because the naturally occurring distribution of deuterium is about0.0156%, about 0.0156% of molecules in a sample synthesized usingnon-enriched starting materials will have deuterium at a given position.

The term “isotopic enrichment factor” refers to the ratio between theisotopic composition and the natural isotopic composition of a specifiedisotope.

It should also be noted that an isotopologue of Compound A can containunnatural proportions of atomic isotopes at one or more of the atoms.For example, an isotopologue of Compound A may be radiolabeled at onemore positions with radioactive isotopes, such as for example tritium(³H), and/or carbon-14 (¹⁴C), or may be isotopically enriched at one ormore positions, such as with deuterium (²H), carbon-13 (¹³C), oxygen-18(¹⁸O) and/or nitrogen-15 (¹⁵N). In certain embodiments, Compound A canbe radiolabeled at one more positions with radioactive isotopes, such asfor example tritium (³H), and/or carbon-14 (¹⁴C), while also beingisotopically enriched at one or more positions, such as with deuterium(²H), carbon-13 (¹³C), oxygen-18 (¹⁸O) and/or nitrogen-15 (¹⁵N).

The term “isotopic composition” refers to the amount of each isotopepresent for a given atom. Radiolabeled and isotopically encrichedcompounds are useful as therapeutic agents, e.g., cancer andinflammation therapeutic agents, research reagents, e.g., binding assayreagents, and diagnostic agents, e.g., in vivo imaging agents. Allisotopic variations of Compound A, whether radioactive or not, areintended to be encompassed within the scope of the embodiments providedherein. In some embodiments, there are provided isotopologues ofCompound A, for example, the isotopologues are deuterium, carbon-13, ornitrogen-15 enriched Compound A.

With regard to the compounds provided herein, when a particular atomicposition is designated as having deuterium or “D,” it is understood thatthe abundance of deuterium at that position is substantially greaterthan the natural abundance of deuterium, which is about 0.0156%. Aposition designated as having deuterium typically has a minimum isotopicenrichment factor of, in particular embodiments, at least 100 (1.56%deuterium incorporation), at least 500 (7.8% deuterium incorporation),at least 1000 (15.6% deuterium incorporation), at least 2000 (31.2%deuterium incorporation), at least 3000 (46.8% deuterium incorporation),at least 3500 (54.6% deuterium incorporation), at least 4000 (62.4%deuterium incorporation), at least 4500 (70.2% deuterium incorporation),at least 5000 (78% deuterium incorporation), at least 5500 (85.8%deuterium incorporation), at least 6000 (93.6% deuterium incorporation),at least 6089.7 (95% deuterium incorporation), at least 6217.9 (97%deuterium incorporation), at least 6346.2 (99% deuterium incorporation),or at least 6378.2 (99.5% deuterium incorporation) at each designateddeuterium atom.

The isotopic enrichment and isotopic enrichment factor of the compoundsprovided herein can be determined using conventional analytical methodsknown to one of ordinary skill in the art, including mass spectrometryand nuclear magnetic resonance spectroscopy.

As used herein, the term “pharmaceutically acceptable salt(s)” refers toa salt prepared from a pharmaceutically acceptable non-toxic acid orbase including an inorganic acid and base and an organic acid and base.Suitable pharmaceutically acceptable base addition salts include, butare not limited to metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made from lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitablenon-toxic acids include, but are not limited to, inorganic and organicacids such as acetic, alginic, anthranilic, L-asparate, benzenesulfonic,benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric,furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic,hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic,phosphoric, propionic, salicylic, stearic, succinic, sulfanilic,sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific non-toxicacids include hydrochloric, hydrobromic, phosphoric, sulfuric, andmethanesulfonic acids. Examples of specific salts thus includehydrochloride and mesylate salts. Others are well-known in the art, seefor example, Remington's Pharmaceutical Sciences, 18th eds., MackPublishing, Easton Pa. (1990) or Remington: The Science and Practice ofPharmacy, 19th eds., Mack Publishing, Easton Pa. (1995).

As used herein and unless otherwise indicated, the term “stereoisomer”or “stereomerically pure” means one stereoisomer of a compound that issubstantially free of other stereoisomers of that compound. For example,a stereomerically pure compound having one chiral center will besubstantially free of the opposite enantiomer of the compound. Astereomerically pure compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, greater than about 90% by weight ofone stereoisomer of the compound and less than about 10% by weight ofthe other stereoisomers of the compound, greater than about 95% byweight of one stereoisomer of the compound and less than about 5% byweight of the other stereoisomers of the compound, or greater than about97% by weight of one stereoisomer of the compound and less than about 3%by weight of the other stereoisomers of the compound. Compounds can havechiral centers and can occur as racemates, individual enantiomers ordiastereomers, and mixtures thereof. All such isomeric forms areincluded within the embodiments disclosed herein, including mixturesthereof. The use of stereomerically pure forms of such compounds, aswell as the use of mixtures of those forms are encompassed by theembodiments disclosed herein. For example, mixtures comprising equal orunequal amounts of the enantiomers of a particular compound may be usedin methods and compositions disclosed herein. These isomers may beasymmetrically synthesized or resolved using standard techniques such aschiral columns or chiral resolving agents. See, e.g., Jacques, J., etal., Enantiomers, Racemates and Resolutions (Wiley Interscience, NewYork, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw Hill, N.Y., 1962); andWilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268(E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972).

It should also be noted the compounds can include E and Z isomers, or amixture thereof, and cis and trans isomers or a mixture thereof. Incertain embodiments, compounds are isolated as either the cis or transisomer. In other embodiments, compounds are a mixture of the cis andtrans isomers.

As used herein, and in the specification and the accompanying claims,the indefinite articles “a” and “an” and the definite article “the”include plural as well as single referents, unless the context clearlyindicates otherwise.

As used herein, and unless otherwise specified, the terms “about” and“approximately,” when used in connection with doses, amounts, or weightpercent of ingredients of a composition or a dosage form, mean a dose,amount, or weight percent that is recognized by one of ordinary skill inthe art to provide a pharmacological effect equivalent to that obtainedfrom the specified dose, amount, or weight percent. In certainembodiments, the terms “about” and “approximately,” when used in thiscontext, contemplate a dose, amount, or weight percent within 30%,within 20%, within 15%, within 10%, or within 5%, of the specified dose,amount, or weight percent.

As used herein, and unless otherwise specified, a crystalline that is“pure,” i.e., substantially free of other crystalline or amorphoussolids, contains less than about 10% by weight of one or more othercrystalline or amorphous solids, less than about 5% by weight of one ormore other crystalline or amorphous solids, less than about 3% by weightof one or more other crystalline or amorphous solids, or less than about1% by weight of one or more other crystalline or amorphous solids.

As used herein, and unless otherwise specified, a solid form that is“substantially physically pure” is substantially free from other solidforms. In certain embodiments, a crystal form that is substantiallyphysically pure contains less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or 0.01% of one or moreother solid forms on a weight basis. The detection of other solid formscan be accomplished by any method apparent to a person of ordinary skillin the art, including, but not limited to, diffraction analysis, thermalanalysis, elemental combustion analysis and/or spectroscopic analysis.

As used herein, and unless otherwise specified, a solid form that is“substantially chemically pure” is substantially free from otherchemical compounds (i.e., chemical impurities). In certain embodiments,a solid form that is substantially chemically pure contains less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%,0.1%, 0.05%, or 0.01% of one or more other chemical compounds on aweight basis. The detection of other chemical compounds can beaccomplished by any method apparent to a person of ordinary skill in theart, including, but not limited to, methods of chemical analysis, suchas, e.g., mass spectrometry analysis, spectroscopic analysis, thermalanalysis, elemental combustion analysis and/or chromatographic analysis.

As used herein, and unless otherwise indicated, a chemical compound,solid form, or composition that is “substantially free” of anotherchemical compound, solid form, or composition means that the compound,solid form, or composition contains, in certain embodiments, less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%0.1%, 0.05%, or 0.01% by weight of the other compound, solid form, orcomposition.

Unless otherwise specified, the terms “solvate” and “solvated,” as usedherein, refer to a solid form of a substance which contains solvent. Theterms “hydrate” and “hydrated” refer to a solvate wherein the solvent iswater. “Polymorphs of solvates” refer to the existence of more than onesolid form for a particular solvate composition. Similarly, “polymorphsof hydrates” refer to the existence of more than one solid form for aparticular hydrate composition. The term “desolvated solvate,” as usedherein, refers to a solid form of a substance which can be made byremoving the solvent from a solvate. The terms “solvate” and “solvated,”as used herein, can also refer to a solvate of a salt, cocrystal, ormolecular complex. The terms “hydrate” and “hydrated,” as used herein,can also refer to a hydrate of a salt, cocrystal, or molecular complex.

“Tautomers” refers to isomeric forms of a compound that are inequilibrium with each other. The concentrations of the isomeric formswill depend on the environment the compound is found in and may bedifferent depending upon, for example, whether the compound is a solidor is in an organic or aqueous solution. For example, in aqueoussolution, pyrazoles may exhibit the following isomeric forms, which arereferred to as tautomers of each other:

As readily understood by one skilled in the art, a wide variety offunctional groups and other structures may exhibit tautomerism and alltautomers of the isotopologues of Compound A are within the scope of thepresent invention.

Unless otherwise specified, the term “composition” as used herein isintended to encompass a product comprising the specified ingredient(s)(and in the specified amount(s), if indicated), as well as any productwhich results, directly or indirectly, from combination of the specifiedingredient(s) in the specified amount(s). By “pharmaceuticallyacceptable,” it is meant a diluent, excipient, or carrier in aformulation must be compatible with the other ingredient(s) of theformulation and not deleterious to the recipient thereof.

“JNK” means a protein or an isoform thereof expressed by a JNK1, JNK2,or JNK3 gene (Gupta, S., Barrett, T., Whitmarsh, A. J., Cavanagh, J.,Sluss, H. K., Derijard, B. and Davis, R. J. The EMBO J., Vol. 15, pp.2760-70 (1996)).

“Treating” as used herein, means an alleviation, in whole or in part, ofa disorder, disease or condition, or one or more of the symptomsassociated with a disorder, disease, or condition, or slowing or haltingof further progression or worsening of those symptoms, or alleviating oreradicating the cause(s) of the disorder, disease, or condition itself.In one embodiment, the disorder is a condition treatable or preventableby inhibition of a JNK pathway, as described herein. In anotherembodiment, the disorder is selected from interstitial pulmonaryfibrosis, systemic sclerosis, scleroderma, chronic allograftnephropathy, antibody mediated rejection, or lupus. In yet anotherembodiment, the disorder is a liver fibrotic disorder, or diabetesand/or metabolic syndrome leading to liver fibrotic disorders, asdescribed herein. In some embodiments, the disorder is a liver fibroticdisorder, such as non-alcoholic steatohepatitis, steatosis (i.e., fattyliver), cirrhosis, primary sclerosing cholangitis, primary biliarycirrhosis, hepatitis, hepatocellular carcinoma, or liver fibrosiscoincident with chronic or repeated alcohol ingestion (alcoholichepatitis), with infection (e.g., viral infection such as HCV), withliver transplant, or with drug induced liver injury (e.g., acetaminophentoxicity). In some embodiments, “treating” means an alleviation, inwhole or in part, of a disorder, disease or condition, or symptomsassociated with diabetes or metabolic syndrome leading to liver fibroticdisorders, such as non-alcoholic steatohepatitis, steatosis (i.e., fattyliver), hepatitis or cirrhosis, or a slowing, or halting of furtherprogression or worsening of those symptoms. In one embodiment, thesymptom is jaundice.

“Preventing” as used herein, means a method of delaying and/orprecluding the onset, recurrence or spread, in whole or in part, of adisorder, disease or condition; barring a subject from acquiring adisorder, disease, or condition; or reducing a subject's risk ofacquiring a disorder, disease, or condition. In one embodiment, thedisorder is a condition treatable or preventable by inhibition of a JNKpathway, as described herein. In another embodiment, the disorder isselected from interstitial pulmonary fibrosis, systemic sclerosis,scleroderma, chronic allograft nephropathy, antibody mediated rejection,or lupus. In one embodiment, the disorder is a liver fibrotic disorder,or diabetes or metabolic syndrome leading to liver fibrotic disorders,as described herein, or symptoms thereof.

The term “effective amount” in connection with an isotopologue ofCompound A means an amount capable of treating or preventing a disorder,disease or condition, or symptoms thereof, disclosed herein.

“Patient” or “subject” is defined herein to include animals, such asmammals, including, but not limited to, primates (e.g., humans), cows,sheep, goats, horses, dogs, cats, rabbits, rats, mice, monkeys,chickens, turkeys, quails, or guinea pigs and the like, in oneembodiment a mammal, in another embodiment a human. In one embodiment, asubject is a human having or at risk for having interstitial pulmonaryfibrosis, systemic sclerosis, scleroderma, chronic allograftnephropathy, antibody mediated rejection, or lupus. In another, asubject is a human having or at risk for having liver fibrotic disordersor diabetes or metabolic syndrome leading to liver fibrotic disorders,or a condition, treatable or preventable by inhibition of a JNK pathway,or a symptom thereof. In one embodiment, a subject is fasted. In anotherembodiment, a subject is fed.

4.1 Compounds

Provided herein are isotopically enriched compounds, includingisotopically enriched Compound A and synthetic intermediates thereof.

Isotopic enrichment (e.g., deuteration) of pharmaceuticals to improvepharmacokinetics (“PK”), pharmacodynamics (“PD”), and toxicity profileshas been demonstrated previously with some classes of drugs. (See, e.g.,Lijinsky et. al., Food Cosmet. Toxicol., Vol. 20, p. 393 (1982);Lijinsky et. al., J. Nat. Cancer Inst., Vol. 69, p. 1127 (1982); Mangoldet. al., Mutation Res. Vol. 308, p. 33 (1994); Gordon et. al., DrugMetab. Dispos., Vol. 15, p. 589 (1987); Zello et. al., Metabolism, Vol.43, p. 487 (1994); Gately et. al., J. Nucl. Med., Vol. 27, p. 388(1986); Wade D, Chem. Biol. Interact., Vol. 117, p. 191 (1999)).

Without being limited by a particular theory, isotopic enrichment of adrug can be used, for example, to (1) reduce or eliminate unwantedmetabolites, (2) increase the half-life of the parent drug, (3) decreasethe number of doses needed to achieve a desired effect, (4) decrease theamount of a dose necessary to achieve a desired effect, (5) increase theformation of active metabolites, if any are formed, and/or (6) decreasethe production of deleterious metabolites in specific tissues and/orcreate a more effective drug and/or a safer drug for combinationtherapy, whether the combination therapy is intentional or not.

Replacement of an atom for one of its isotopes may often result in achange in the reaction rate of a chemical reaction or an enzymecatalyzed reaction. This phenomenon is known as the Kinetic IsotopeEffect (“KIE”). For example, if a C—H bond is broken during arate-determining step in a chemical reaction (i.e., the step with thehighest transition state energy), substitution of a deuterium for thathydrogen can cause a decrease in the reaction rate and the process mayslow down. This phenomenon is known as the Deuterium Kinetic IsotopeEffect (“DKIE”). (See, e.g, Foster et al., Adv. Drug Res., vol. 14, pp.1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol., vol. 77, pp.79-88 (1999)).

The magnitude of the DKIE can be expressed as the ratio between therates of a given reaction in which a C—H bond is broken, and the samereaction where deuterium is substituted for hydrogen. The DKIE can rangefrom about 1 (no isotope effect) to very large numbers, such as 50 ormore, meaning that the reaction can be fifty, or more, times slower whendeuterium is substituted for hydrogen. Without being limited by aparticular theory, high DKIE values may be due in part to a phenomenonknown as tunneling, which is a consequence of the uncertainty principle.Tunneling is ascribed to the small mass of a hydrogen atom, and occursbecause transition states involving a proton can sometimes form in theabsence of the required activation energy. Because deuterium has moremass than hydrogen, it statistically has a much lower probability ofundergoing this phenomenon.

Tritium (“T”) is a radioactive isotope of hydrogen, used in research,fusion reactors, neutron generators and radiopharmaceuticals. Tritium isa hydrogen atom that has 2 neutrons in the nucleus and has an atomicweight close to 3. It occurs naturally in the environment in very lowconcentrations, most commonly found as T₂O. Tritium decays slowly(half-life=12.3 years) and emits a low energy beta particle that cannotpenetrate the outer layer of human skin. Internal exposure is the mainhazard associated with this isotope, yet it must be ingested in largeamounts to pose a significant health risk. As compared with deuterium, alesser amount of tritium must be consumed before it reaches a hazardouslevel. Substitution of tritium (“T”) for hydrogen results in yet astronger bond than deuterium and gives numerically larger isotopeeffects. Similarly, substitution of isotopes for other elements,including, but not limited to, ¹³C or ¹⁴C for carbon, ³³S, ³⁴S, or ³⁶Sfor sulfur, ¹⁵N for nitrogen, and ¹⁷O or ¹⁸O for oxygen, may lead to asimilar kinetic isotope effect.

The animal body expresses a variety of enzymes for the purpose ofeliminating foreign substances, such as therapeutic agents, from itscirculation system. Examples of such enzymes include the cytochrome P450enzymes (“CYPs”), esterases, proteases, reductases, dehydrogenases, andmonoamine oxidases, to react with and convert these foreign substancesto more polar intermediates or metabolites for renal excretion. Some ofthe most common metabolic reactions of pharmaceutical compounds involvethe oxidation of a carbon-hydrogen (C—H) bond to either a carbon-oxygen(C—O) or carbon-carbon (C—C) pi-bond. The resultant metabolites may bestable or unstable under physiological conditions, and can havesubstantially different pharmacokinetic, pharmacodynamic, and acute andlong-term toxicity profiles relative to the parent compounds. For manydrugs, such oxidations are rapid. These drugs therefore often requirethe administration of multiple or high daily doses.

Therefore, isotopic enrichment at certain positions of a compoundprovided herein may produce a detectable KIE that affects thepharmacokinetic, pharmacologic, and/or toxicological profiles of acompound provided herein in comparison with a similar compound having anatural isotopic composition. In one embodiment, the deuteriumenrichment is performed on the site of C—H bond cleavage duringmetabolism.

In some embodiments, provided herein are deuterated analogues ofCompound A, wherein one or more atomic positions of Compound A is/areisotopically enriched with deuterium. Certain embodiments herein providecompounds of the following structure of Compound A:

wherein one or more Y atoms (i.e., Y¹, Y², Y³, Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹,Y¹⁰, Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵, Y¹⁶, Y¹⁷, Y¹⁸, Y¹⁹, Y²⁰, Y²¹, and Y²²)is/are hydrogen(s) isotopically enriched with deuterium, and anyremaining Y atom(s) is/are non-enriched hydrogen atom(s). In particularembodiments, one, two, three, four, five, six, seven, eight or nine ofthe indicated Y atoms is/are isotopically enriched with deuterium, andany remaining Y atom(s) is/are non-enriched hydrogen(s). In oneembodiment, all of Y¹, Y², Y³, Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹²,Y¹³, Y¹⁴, Y¹⁵, Y¹⁶, Y¹⁷, Y¹⁸, Y¹⁹, Y²⁰, Y²¹, and Y²² are isotopicallyenriched with deuterium.

In certain embodiments, one or more Y atoms on the cyclohexyl portion ofCompound A is/are deuterium-enriched. For example, particular compoundsprovided herein include the following listed compounds, wherein thelabel “D” indicates a deuterium-enriched atomic position, i.e., a samplecomprising the given compound has a deuterium enrichment at theindicated position(s) above the natural abundance of deuterium:

In certain embodiments, one or more Y atoms on the tert-butyl portion ofCompound A are deuterium-enriched. For example, particular compoundsprovided herein include, but are not limited to, the following listedcompounds, wherein the label “D” indicates a deuterium-enriched atomicposition, i.e., a sample comprising the given compound has a deuteriumenrichment at the indicated position(s) above the natural abundance ofdeuterium:

In certain embodiments, one or more Y atoms on the pyrimidine portion ofCompound A is/are deuterium-enriched. For example, particular compoundsprovided herein include, but are not limited to, the following listedcompounds, wherein the label “D” indicates a deuterium-enriched atomicposition, i.e., a sample comprising the given compound has a deuteriumenrichment at the indicated position(s) above the natural abundance ofdeuterium:

In certain embodiments, one or more Y atoms on the cyclohexyl,tert-butyl, and/or pyrimidine portions of Compound A is/aredeuterium-enriched, i.e., any combination of deuteration enrichmentshown above is encompassed. In some embodiments the compound is selectedfrom:

In one embodiment, the compound is

In certain embodiments, the isotopologue examples above have thestereochemistry of the following structure:

It is understood that one or more deuteriums may exchange with hydrogenunder physiological conditions.

4.2 Synthesis

The compounds described herein may be synthesized using methods known tothose of ordinary skill in the art. For example, particular compoundsdescribed herein are synthesized using standard synthetic organicchemistry techniques known to those of ordinary skill in the art.

In some embodiments, known procedures for the synthesis of Compound 1are employed, wherein one or more of the reagents, starting materials,precursors, or intermediates are replaced by one or moreisotopically-enriched reagents or intermediates, including but notlimited to one or more deuterium-enriched reagents, starting materials,precursors, or intermediates. Such known procedures for the synthesis ofCompound 1 and tautomers thereof include, but are not limited to, thosedescribed in U.S. Patent Application Publication No. 2013/0029987,published on Jan. 31, 2013, and International Pub. No. WO2012/145569,the entireties of each of which are incorporated by reference herein.Isotopically enriched reagents, starting materials, precursors, andintermediates are commercially available or may be prepared by routinechemical reactions known to one of skill in the art.

U.S. Patent Application Publication No. 2013/0029987 describedprocedures for synthesizing Compound 1 as shown in the Scheme 1 below.

In some embodiments, one or more hydrogen positions of the cyclohexyl,tert-butyl, and/or pyrimidine portions of Compound 1 are enriched withdeuterium through organic synthesis. In some embodiments, the methods ofScheme 1 are employed.

In particular embodiments, the methods of Scheme 2 are employed, asdepicted below:

In certain embodiments, the methods of Scheme 2 have the stereochemistryas depicted below in Scheme 3:

In some embodiments, the methods of Scheme 2 are employed, wherein oneor more deuterium-enriched precursors are used, as depicted below inScheme 4:

wherein one or more Y atoms (i.e., Y¹⁻²²) is/are hydrogen(s)isotopically enriched with deuterium, and any remaining Y atom(s) is/arenon-enriched hydrogen atom(s). In particular embodiments, one, two,three, four, five, six, seven, eight, nine or more of the indicated Yatoms is/are isotopically enriched with deuterium, and any remaining Yatom(s) is/are non-enriched hydrogen(s). Isotopically enriched reagents,starting materials, and/or precursors may be obtained commercially orthrough techniques known to those of skill in the art.

In certain embodiments, the methods of Scheme 4 have the stereochemistryof the following Scheme 5:

wherein one or more Y atoms (i.e., Y¹⁻²²) is/are hydrogen(s)isotopically enriched with deuterium, and any remaining Y atom(s) is/arenon-enriched hydrogen atom(s). In particular embodiments, one, two,three, four, five, six, seven, eight, nine or more of the indicated Yatoms is/are isotopically enriched with deuterium, and any remaining Yatom(s) is/are non-enriched hydrogen(s). Isotopically enriched reagents,starting materials, and/or precursors may be obtained commercially orthrough techniques known to those of skill in the art.

In certain embodiments, the following isotopically enriched startingmaterials can be used in the methods described herein to affordadditional isotopologues of Compound A.

In certain embodiments, a deuterium-enriched analog of precursor (5) issynthesized, wherein one of the Y atoms (i.e., Y¹) is hydrogen(s)isotopically enriched with deuterium, and any remaining Y atom(s) is/arenon-enriched hydrogen atom(s). In particular embodiments, one of theindicated Y atoms is/are isotopically enriched with deuterium, and anyremaining Y atom(s) is/are non-enriched hydrogen(s). Isotopicallyenriched reagents, starting materials, and/or precursors may be obtainedcommercially or through techniques known to those of skill in the art.

A deuterium-enriched pyrimidine precursor (5) for deuterium-enrichedisotopologues of Compound A can be prepared by methods known in the art.For example, as shown in Scheme 6 above,2,4-dichloropyrimidine-5-carboxamide-²H (5) can be prepared by combiningdiethyl malonate (14), triethoxymethane-²H (15), and urea to generatediethyl 2-(ureidomethylene)malonate-²H (16). Diethyl2-(ureidomethylene)malonate-²H (16) is combined with sodium methoxide toafford ethyl 2,4-dihydroxypyrimidine-5-carboxylate-²H (17). Treatment ofethyl 2,4-dihydroxypyrimidine-5-carboxylate-²H (17) with hydrochloricacid followed by halogenation of the resulting2,4-dihydroxypyrimidine-5-carboxylic acid (18) with phosphoryl chlorideand triethylamine affords 2,4-dichloropyrimidine-5-carboxylic acid-²H(19). To a solution of 2,4-dichloropyrimidine-5-carboxylic acid-²H (19)is added thionyl chloride and ammonia to afford the deuterium-enrichedprecursor 2,4-dichloropyrimidine-5-carboxamide (5), which can beincorporated by the synthesis methods described herein.

In certain embodiments, a deuterium-enriched analog of precursor (6) issynthesized, wherein one or more Y atoms (i.e., Y¹¹⁻²²) is/arehydrogen(s) isotopically enriched with deuterium, and any remaining Yatom(s) is/are non-enriched hydrogen atom(s). In particular embodiments,one of the indicated Y atoms is/are isotopically enriched withdeuterium, and any remaining Y atom(s) is/are non-enriched hydrogen(s).Isotopically enriched reagents, starting materials, and/or precursorsmay be obtained commercially or through techniques known to those ofskill in the art.

A deuterium-enriched cyclohexyl precursor (6) for deuterium-enrichedisotopologues of Compound A can be prepared by methods known in the art.For example, as shown in Scheme 7 above,(1R,2R,5R)-5-amino-2-methylcyclohexanol hydrochloride ²H (6) can beprepared via an asymmetric Diels-Alder reaction by combining2,2,2-trifluoroethyl acrylate-²H (8) and isoprene ²H (9) in the presenceof borane and sulfonamide catalysts to afford (R)-2,2,2-trifluoroethyl4-methylcyclohex-3-enecarboxylate-²H (10). A solution of(R)-2,2,2-trifluoroethyl 4-methylcyclohex-3-enecarboxylate-²H (10) inmethanol and water is treated with lithium hydroxide to afford(R)-4-methylcyclohex-3-enecarboxylic acid-²H (11) with retention ofstereochemistry. Treatment of (R)-4-methylcyclohex-3-enecarboxylicacid-²H (11) with diphenylphosphoryl azide (DPPA) and triethylamine,followed by tert-butoxide and copper chloride, affords (R)-tert-butyl(4-methylcyclohex-3-en-1-yl)carbamate-²H (12). Hydroboration withdiisopinocampheylborane-²H catalyst (21) followed by oxidation of thealkene of (R)-tert-butyl (4-methylcyclohex-3-en-1-yl)carbamate-²H (12)provides tert-butyl ((1R,3R,4R)-3-hydroxy-4-methylcyclohexyl)carbamate(13). Treatment of tert-butyl((1R,3R,4R)-3-hydroxy-4-methylcyclohexyl)carbamate (13) withhydrochloric acid affords the deuterium-enriched precursor(1R,2R,5R)-5-amino-2-methylcyclohexanol hydrochloride (6), which can beincorporated by the synthesis methods described herein.

In certain embodiments, partial deuteration of a deuterium-enrichedprecursor (6) for deuterium-enriched isotopologues of Compound A can beprepared. For example, as shown below in reaction Schemes 8 to 14,(1R,2R,5R)-5-amino-2-methylcyclohexanol hydrochloride-²H (6) can beprepared via an asymmetric Diels-Alder reaction by combining differentdeuterated or undeuterated combinations of 2,2,2-trifluoroethylacrylate-²H (8) and isoprene ²H (9) to afford (R)-2,2,2-trifluoroethyl4-methylcyclohex-3-enecarboxylate-²H (10). Treatment of the resulting(R)-2,2,2-trifluoroethyl 4-methylcyclohex-3-enecarboxylate-²H (10) withdifferent deuterated or undeuterated combinations ofdiisopinocampheylborane-²H catalyst (21) affords the deuterium-enrichedprecursor (1R,2R,5R)-5-amino-2-methylcyclohexanol hydrochloride (6),which can be incorporated by the synthesis methods described herein.

In certain embodiments, a deuterium-enriched acrylate precursor (8) fordeuterium-enriched isotopologues of Compound A can be prepared bymethods known in the art (Manufacture of fluoroalkyl (meth)acrylateswith high yield in presence of polyphosphoric acid, Tani, Teruo; Ito,Keisuke; Imamura, Mitsunobu; Kawakami, Naohiko, Jpn. Kokai Tokkyo Koho,2005225774). For example, as shown in Scheme 15 below, condensation ofperdeuterio-acrylic acid (20) with 2,2,2-trifluoroethanol in thepresence of polyphosphoric acid provides 2,2,2-trifluoroethylacrylate-²H (8).

In certain embodiments, one or more hydrogen positions of precursors (8)and (9) of the cyclohexyl portion of Compound A are enriched withdeuterium through organic synthesis. For example, a deuterium-enrichedprecursor (9) for deuterium-enriched isotopologues of Compound A can beprepared by methods known in the art (Craig, David; Regenass, Franz A.;Fowler, Raymond B., J. of Organic Chemistry, February 1959, Vol. 24, No.2, 240-244).

In certain embodiments, partial deuteration of precursor (9) fordeuterium-enriched isotopologues of Compound A can be prepared bymethods known in the art (Sato, Hisaya; Ono, Akihisha; Tanaka, Yasuyuki,Polymer, June 1977, Vol. 18, No. 6, 580-586).

In certain embodiments, diisopinocampheylborane-²H catalyst (21) fordeuterium-enriched isotopologues of Compound A can be prepared bymethods known in the art (Wolfe, Saul; Rauk, Arvi, Canadian J. ofChemistry, 1966, Vol. 44, No. 21, 2591-93).

In certain embodiments, partial deuteration of deuterium-enrichedisotopologues of Compound A can be prepared. For example, as shown belowin reaction Schemes 16 to 24, isotopologues of Compound A can beprepared by combining different deuterated or undeuterated combinationsof 2,4-dichloropyrimidine-5-carboxamide-²H (5) and(1R,2R,5R)-5-amino-2-methylcyclohexanol hydrochloride-²H (6) in thepresence of potassium carbonate to afford2-chloro-4-((3-hydroxy-4-methylcyclohexyl)amino)pyrimidine-5-carboxamide-²H(7). Treatment of2-chloro-4-((3-hydroxy-4-methylcyclohexyl)amino)pyrimidine-5-carboxamide-²H(7) with deuterated or undeuterated tert-butyl amine affords adeuterium-enriched isotopologue of Compound A.

In certain embodiments, the methods described in Scheme 1 are employed.In particular embodiments, the methods of Scheme 1 are employed, whereindeuterium-enriched reagents are used, similar to above.

In certain embodiments, one or more hydrogen positions of the pyrimidineportion of Compound A is/are deuterated by subjecting Compound A toconditions suitable for aromatic deuteration, which are known in theart, including for example, those disclosed in the following references,each of which are incorporated herein by reference in their entireties:U.S. Publication No. 2007/0255076; March, J. “Advanced OrganicChemistry, Reactions, Mechanisms, and Structure,” Fourth Ed., Wiley, NewYork, 1992; Larsen et al., J. Org. Chem., 43(18), 3602, 1978; Blake etal., J. Chem. Soc., Chem Commun., 930, 1975; and references citedtherein. For example, as depicted in Scheme 25 below, Compound A istreated with D₂O over 5% Pt/C under hydrogen gas to provide anisotopologue of Compound A, as depicted in the scheme above. In certainembodiments, Compound A is converted to a Compound A derivative (e.g.,by incorporation of a protecting group), subjected to aromaticdeuteration conditions, and converted to deuterium-enriched Compound A.

4.3 Methods of Use

Provided herein are methods of treating, preventing, and/or managingvarious diseases or disorders using isotopologues of Compound A asprovided herein, or a pharmaceutically acceptable salt, solvate (e.g.,hydrate), prodrug, clathrate, or stereoisomer thereof.

Pharmaceutical compositions and dosage forms of the isotopologues ofCompound A have utility as pharmaceuticals to treat, prevent or improveconditions in animals or humans. The isotopologues of Compound A areactive against protein kinases, particularly JNK1 and/or JNK2.Accordingly, provided herein are many uses of pharmaceuticalcompositions and dosage forms of the isotopologues of Compound A,including the treatment or prevention of those diseases set forth below.The methods provided herein comprise the administration of apharmaceutical composition or dosage form of an isotopologue of CompoundA to a subject in need thereof. In one aspect, provided herein aremethods of inhibiting a kinase in a cell expressing said kinase,comprising contacting said cell with an effective amount of apharmaceutical composition or dosage form of an isotopologue of CompoundA. In one embodiment, the kinase is JNK1, JNK2, or a mutant or isoformthereof, or a combination thereof.

In another aspect, provided herein are methods for treating orpreventing one or more disorders selected from interstitial pulmonaryfibrosis, systemic sclerosis, scleroderma, chronic allograftnephropathy, antibody mediated rejection, or lupus, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition or dosage form of an isotopologue of CompoundA. In some such embodiments, the lupus is lupus erythematosus (such asdiscoid lupus erythematosus, or cutaneous lupus erythematosus) orsystemic lupus.

In another aspect, provided herein are methods for treating orpreventing liver fibrotic disorders, such as non-alcoholicsteatohepatitis, steatosis (i.e. fatty liver), cirrhosis, primarysclerosing cholangitis, primary biliary cirrhosis, hepatitis,hepatocellular carcinoma, and liver fibrosis coincident with chronic orrepeated alcohol ingestion (alcoholic hepatitis), with infection (e.g.,viral infection such as HCV), with liver transplant, or with druginduced liver injury (e.g., acetaminophen toxicity), comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition or dosage form of an isotopologue of CompoundA. In some such aspects, provided herein are methods for treating orpreventing diabetes or metabolic syndrome leading to liver fibroticdisorders, such as non-alcoholic steatohepatitis, steatosis (i.e. fattyliver), cirrhosis, primary sclerosing cholangitis, primary biliarycirrhosis, and hepatitis, comprising administering to a subject in needthereof an effective amount of a pharmaceutical composition or dosageform of an isotopologue of Compound A.

In another aspect, provided herein are methods for treating orpreventing conditions treatable or preventable by inhibition of JNK1and/or JNK2, the method comprising administering to a subject in needthereof an effective amount of a pharmaceutical composition or dosageform of an isotopologue of Compound A. Examples of such conditionsinclude rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis,asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonarydisease, cystic fibrosis, inflammatory bowel disease, irritable bowelsyndrome, mucous colitis, ulcerative colitis, Crohn's disease,Huntington's disease, hepatitis, pancreatitis, nephritis, multiplesclerosis, lupus erythematosus, Type II diabetes, obesity,atherosclerosis, restenosis following angioplasty, left ventricularhypertrophy, myocardial infarction, stroke, ischemic damages of heart,lung, gut, kidney, liver, pancreas, spleen and brain, acute or chronicorgan transplant rejection, preservation of the organ fortransplantation, organ failure or loss of limb (e.g., including, but notlimited to, that resulting from ischemia-reperfusion injury, trauma,gross bodily injury, car accident, crush injury or transplant failure),graft versus host disease, endotoxin shock, multiple organ failure,psoriasis, burn from exposure to fire, chemicals or radiation, eczema,dermatitis, skin graft, ischemia, ischemic conditions associated withsurgery or traumatic injury (e.g., vehicle accident, gunshot wound orlimb crush), epilepsy, Alzheimer's disease, Parkinson's disease,immunological response to bacterial or viral infection, cachexia,angiogenic and proliferative diseases, solid tumor, and cancers of avariety of tissues such as colon, rectum, prostate, liver, lung,bronchus, pancreas, brain, head, neck, stomach, skin, kidney, cervix,blood, larynx, esophagus, mouth, pharynx, urinary bladder, ovary oruterine.

4.4 Routes of Administrations and Dosage

Pharmaceutical compositions and dosage forms of the isotopologues ofCompound A can be administered to a subject orally, topically orparenterally in the conventional form of preparations, such as tablets,granules, powder, troches, pills, suppositories, injections,suspensions, syrups, patches, creams, lotions, ointments, gels, sprays,solutions and emulsions. The effective amount of an isotopologue ofCompound A in the pharmaceutical composition may be at a level that willexercise the desired effect; for example, about 0.005 mg/kg of asubject's body weight to about 10 mg/kg of a subject's body weight inunit dosage for both oral and parenteral administration.

The dose of an isotopologue of Compound A to be administered to asubject is rather widely variable and can be subject to the judgment ofa healthcare practitioner. In general, an isotopologue of Compound A canbe administered one to four times a day in a dose of about 0.005 mg/kgof a subject's body weight to about 10 mg/kg of a subject's body weightin a subject, but the above dosage may be properly varied depending onthe age, body weight and medical condition of the subject and the typeof administration. In one embodiment, the dose is about 0.01 mg/kg of asubject's body weight to about 5 mg/kg of a subject's body weight, about0.05 mg/kg of a subject's body weight to about 1 mg/kg of a subject'sbody weight, about 0.1 mg/kg of a subject's body weight to about 0.75mg/kg of a subject's body weight or about 0.25 mg/kg of a subject's bodyweight to about 0.5 mg/kg of a subject's body weight. In one embodiment,one dose is given per day. In any given case, the amount of anisotopologue of Compound A administered will depend on such factors asthe solubility of the active component, the formulation used and theroute of administration. In one embodiment, application of a topicalconcentration provides intracellular exposures or concentrations ofabout 0.01-10 μM.

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder comprising the administration of apharmaceutical composition or dosage form comprising about 0.375 mg/dayto about 750 mg/day, about 0.75 mg/day to about 375 mg/day, about 3.75mg/day to about 75 mg/day, about 7.5 mg/day to about 55 mg/day or about18 mg/day to about 37 mg/day of an isotopologue of Compound A to asubject in need thereof.

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder comprising the administration of apharmaceutical composition or dosage form comprising about 1 mg/day toabout 1200 mg/day, about 10 mg/day to about 1200 mg/day, about 100mg/day to about 1200 mg/day, about 400 mg/day to about 1200 mg/day,about 600 mg/day to about 1200 mg/day, about 400 mg/day to about 800mg/day, about 60 mg/day to about 720 mg/day, about 240 mg/day to about720 mg/day or about 600 mg/day to about 800 mg/day of an isotopologue ofCompound A to a subject in need thereof. In a particular embodiment, themethods disclosed herein comprise the administration of a pharmaceuticalcomposition or dosage form comprising about 400 mg/day, about 600 mg/dayor about 800 mg/day of an isotopologue of Compound A to a subject inneed thereof.

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder comprising the administration of apharmaceutical composition or dosage form comprising about 10 mg/day toabout 720 mg/day, about 10 mg/day to about 480 mg/day, about 60 mg/dayto about 720 mg/day or about 240 mg/day to about 720 mg/day of anisotopologue of Compound A to a subject in need thereof. In oneembodiment, provided herein are methods for the treatment or preventionof a disease or disorder comprising the administration of apharmaceutical composition or dosage form comprising about 60 mg/day,about 160 mg/day, or about 400 mg/day of an isotopologue of Compound Ato a subject in need thereof. In another embodiment, provided herein aremethods for the treatment or prevention of a disease or disordercomprising the administration of a pharmaceutical composition or dosageform comprising about 200 mg/day of an isotopologue of Compound A to asubject in need thereof.

In one embodiment, provided herein are methods for the treatment orprevention of a disease or disorder comprising the administration of apharmaceutical composition, or dosage form comprising about 10 mg/day,about 30 mg/day, about 60 mg/day, about 120 mg/day, about 240 mg/day,about 480 mg/day, or about 720 mg/day of Compound A to a subject in needthereof. In one embodiment, provided herein are methods for thetreatment or prevention of a disease or disorder comprising theadministration of a pharmaceutical composition, or dosage formcomprising about 60 mg/day, about 160 mg/day, or about 400 mg/day ofCompound A to a subject in need thereof. In another embodiment, providedherein are methods for the treatment or prevention of a disease ordisorder comprising the administration of a pharmaceutical composition,or dosage form comprising about 200 mg/day of Compound A to a subject inneed thereof. In one embodiment, provided herein are methods for thetreatment or prevention of a disease or disorder comprising theadministration of a pharmaceutical composition, or dosage formcomprising about 10 mg/day, about 30 mg/day, about 60 mg/day, about 120mg/day, about 160 mg/day, about 200 mg/day, about 240 mg/day, about 400mg/day, about 480 mg/day, or about 720 mg/day of Compound A to a subjectin need thereof.

In another embodiment, provided herein are unit dosage formulations thatcomprise between about 10 mg and about 100 mg, about 1 mg and about 200mg, about 30 mg and about 200 mg, about 35 mg and about 1400 mg, about125 mg and about 1000 mg, about 250 mg and about 1000 mg, or about 500mg and about 1000 mg of an isotopologue of Compound A.

In another embodiment, provided herein are unit dosage formulations thatcomprise about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg,about 30 mg, about 35 mg, about 50 mg, about 60 mg, about 70 mg, about100 mg, about 120 mg, about 125 mg, about 140 mg, about 175 mg, about200 mg, about 240 mg, about 250 mg, about 280 mg, about 350 mg, about480 mg, about 500 mg, about 560 mg, about 700 mg, about 720 mg, about750 mg, about 1000 mg or about 1400 mg of an isotopologue of Compound A.

In another embodiment, provided herein are unit dosage formulations thatcomprise about 30 mg, about 100 mg or about 200 mg of an isotopologue ofCompound A.

Pharmaceutical compositions and dosage forms of an isotopologue ofCompound A can be administered once, twice, three, four or more timesdaily. In one embodiment, pharmaceutical compositions and dosage formsof an isotopologue of Compound A can be administered once daily for 14days.

Pharmaceutical compositions and dosage forms of an isotopologue ofCompound A can be administered orally for reasons of convenience. In oneembodiment, when administered orally, pharmaceutical compositions anddosage forms of the isotopologues of Compound A are administered with ameal and water. In another embodiment, pharmaceutical compositions anddosage forms of the isotopologues of Compound A (e.g., granules ordispersible tablets) are dispersed in water or juice (e.g., apple juiceor orange juice) and administered orally as a suspension.

Pharmaceutical compositions and dosage forms of the isotopologues ofCompound A can also be administered intradermally, intramuscularly,intraperitoneally, percutaneously, intravenously, subcutaneously,intranasally, epidurally, sublingually, intracerebrally, intravaginally,transdermally, rectally, mucosally, by inhalation, or topically to theears, nose, eyes, or skin. The mode of administration is left to thediscretion of the health-care practitioner, and can depend in-part uponthe site of the medical condition.

4.5 Process for Making Dosage Forms

Dosage forms provided herein can be prepared by any of the methods ofpharmacy, but all methods include the step of bringing the activeingredient into association with the excipient, which constitutes one ormore necessary ingredients. In general, the compositions are prepared byuniformly admixing (e.g., direct blend) the active ingredient withliquid excipients or finely divided solid excipients or both, and then,if necessary, shaping the product into the desired presentation (e.g.,compaction such as roller-compaction). If desired, tablets can be coatedby standard aqueous or non-aqueous techniques.

A dosage form provided herein can be prepared by compression or molding,optionally with one or more accessory ingredients. Compressed tabletscan be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with an excipient as above and/or a surface active or dispersingagent. Molded tablets can be made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.

In some embodiments, the active ingredients and excipients are directlyblended and compressed directly into tablets. A direct-blended dosageform may be more advantageous than a compacted (e.g., roller-compacted)dosage form in certain instances, since direct-blending can reduce oreliminate the harmful health effects that may be caused by airborneparticles of ingredients during the manufacture using compactionprocess.

Direct blend formulations may be advantageous in certain instancesbecause they require only one blending step, that of the active andexcipients, before being processed into the final dosage form, e.g.,tablet. This can reduce the production of airborne particle or dust to aminimum, while roller-compaction processes may be prone to produce dust.In roller-compaction process, the compacted material is often milledinto smaller particles for further processing. The milling operation canproduce significant amounts of airborne particles, since the purpose forthis step in manufacturing is to reduce the materials particle size. Themilled material is then blended with other ingredients prior tomanufacturing the final dosage form.

For certain active ingredients, in particular for a compound with a lowsolubility, the active ingredient's particle size is reduced to a finepowder in order to help increase the active ingredient's rate ofsolubilization. The increase in the rate of solubilization is oftennecessary for the active ingredient to be effectively absorbed in thegastrointestinal tract. However for fine powders to be directly-blendedand compressed to tablets, the excipients should preferably providecertain characteristics which render the ingredients suitable for thedirect-blend process. Examples of such characteristics include, but arenot limited to, acceptable flow characteristics. In one embodiment,therefore, provided herein is the use of, and compositions comprising,excipients which may provide characteristics, which render the resultingmixture suitable for direct-blend process, e.g., good flowcharacteristics.

In certain embodiments, provided herein are methods for preparing acomposition provided herein, comprising: (i) weighing out the desiredamount of an isotopologue of Compound A and the desired amount of afirst portion of excipients; (ii) preparing an aqueous solution ofsurfactant(s); (iii) passing the mixture of an isotopologue of CompoundA and the first portion of the excipients without the surfactant(s)through a screen; (iv) mixing or blending an isotopologue of Compound A,the aqueous solution of surfactant(s) and the first portion of theexcipients; (v) drying the mixture; (vi) passing a second portion of theexcipients through a screen; (vii) mixing or blending the mixture ofstep (v) and the second portion of the excipients; (viii) weighing outthe desired amount of lubricating agents; (ix) passing the lubricatingagents through a screen; (x) mixing or blending the mixture of step(vii) and the lubricating agents; (xi) compressing the mixture of step(x); and (ix) coating the compressed mixture with a coating agent. Inone embodiment, the mixture of an isotopologue of Compound A, theexcipients and the lubricating agents is compressed into a tablet form.In one embodiment, the screen is 18 mesh screen. In another embodiment,the screen is 1000 μm screen. In one embodiment, the screen is 20 meshscreen. In another embodiment, the screen is 841 μm screen. In oneembodiment, the screen is 30 mesh screen. In another embodiment, thescreen is 595 μm screen.

5 EXAMPLES

General:

Isotopically enriched analogs of the compounds provided herein maygenerally be prepared according known procedures for the synthesis ofCompound A, wherein one or more of the reagents, starting materials,precursors, or intermediates used is replaced by one or moreisotopically enriched reagents, starting materials, precursors, orintermediates. Isotopically enriched reagents, starting materials,precursors, or intermediates are commercially available or may beprepared by routine procedures known to one of skill in the art. Schemesfor the preparation of exemplary isotopically enriched compounds areillustrated below.

Abbreviations

AcOH: acetic acid

AP: area purity

CP: chemical purity

DCM: dichloromethane

DIEA: diisopropylamine

DPPA: diphenylphosphoryl azide

DMSO: dimethylsulfoxide

EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

EtOAc: ethyl acetate

EtOH: ethanol

H₂: hydrogen gas

H²: deuterium

HOtBu: tert-butoxide

HPLC: high performance liquid chromatography

i-PrOH: isopropanol

LC-MS: liquid chromatography/mass spectrometry

MeCN: acetonitrile

MeOH: methanol

MTBE: methyl tert-butyl ether

NMR: nuclear magnetic resonance

PMA: phosphomolybdic acid

Pt/C: platinum on carbon

RCP: radiochemical purity

RT: retention time

TEA: triethylamine

TFA: trifluoroacetic acid

THF: tetrahydrofuran

TLC: thin layer chromatography

Trt: trityl

5.1 Example 14-{[(1R,3R,4R)-3-hydroxy-4-methylcyclohexyl]amino}-2-{[2-(2H3)methyl(2H6)propan-2-yl]amino}pyrimidine-5-carboxamide(Compound A)

As depicted in Scheme 26 below, a mixture of precursor (7) (1.6 g) andt-butyl-d₉-amine (4.95 mL) in DMSO (8 mL) was heated at 65° C. for 91hours. The mixture was cooled to 60° C. Water (8 mL) was charged whilemaintaining the batch temperature at 60° C. After 2.5 hours at 60° C.,water (24 mL) was charged while maintaining the batch temperature at 60°C. After 2 hours at 60° C., the batch was cooled to 25° C. and kept for11 hours. The solids was filtered and washed with water. The solids wasdried in a vacuum oven at 40° C. with nitrogen bleed to give4-{[(1R,3R,4R)-3-hydroxy-4-methylcyclohexyl]amino}-2-{[2-(2H3)methyl(2H6)propan-2-yl]amino}pyrimidine-5-carboxamide(Compound A) (1.72 g). ¹H NMR (DMSO-d₆, 300 MHz): δ 0.94 (d, J=6.4 Hz,3H), 0.96-1.04 (m, 1H), 1.04-1.33 (m, 3H), 1.67 (dd, J=3.3, 13.1 Hz,1H), 1.89 (d, J=11.0 Hz, 1H), 2.12 (d, J=11.1 Hz, 1H), 2.85-3.06 (m,1H), 3.71-4.03 (m, 1H), 4.55 (dd, J=2.1, 5.5 Hz, 1H), 6.59 (br. s., 1H),6.73-8.01 (m, 2H), 8.34 (s, 1H), 8.93 (br. s., 1H).

5.2 Determination of Isotopic Enrichment

Isotopic enrichment may be confirmed and quantified by mass spectrometryand/or NMR, including, for example, proton-NMR; carbon-13 NMR; ornitrogen-15 NMR.

Isotopic enrichment may also be confirmed by single-crystal neutrondiffraction. For example, the isotopic ratio at a particularhydrogen/deuterium position in a deuterated Compound A can be determinedusing single-crystal neutron diffraction. Neutron diffraction isadvantageous because neutrons are scattered by the nucleus of an atom,therefore allowing for discrimination between isotopes, such as hydrogenand deuterium, which differ in the number of neutrons in the nucleus.

A single crystal of suitable size and quality comprising the deuteratedCompound A is grown using standard methods of crystal growth. Forsingle-crystal neutron diffraction experiments, crystals of severalcubic millimeters are generally required for suitable data collection. Aminimum size for a single crystal is typically about 1 cubic millimeter.Suitable single crystals are obtained by dissolving the deuteratedCompound A in a solvent with appreciable solubility, then slowlyevaporating or cooling the solution to yield crystals of suitable sizeand quality. Alternatively, suitable single crystals are obtained bydissolving the deuterated Compound A in a solvent with appreciablesolubility, then slowly diffusing into the solution of antisolvent(i.e., a solvent in which the deuterated Compound A is not appreciablysoluble) to yield crystals of suitable size and quality. These and othersuitable methods of crystal growth are known in the art and aredescribed, e.g., in George H. Stout & Lyle H. Jensen, X-Ray StructureDetermination: A Practical Guide 74-92 (John Wiley & Sons, Inc. 2nd ed.1989) (the entirety of which is incorporated herein).

After isolating a suitable single crystal comprising the deuteratedCompound A, the crystal is mounted in a neutron beam, neutrondiffraction data is collected, and the crystal structure is solved andrefined. Different neutron sources can be used, including steady-statesources and pulsed spallation sources. Examples of steady-state sourcesinclude the Grenoble ILL High Flux Reactor (Grenoble, France) and theOak Ridge High Flux Isotope Reactor (Oak Ridge, Tenn.). Examples ofpulsed spallation sources include ISIS, the spallation neutron source atRutherford Appleton Laboratory (Oxfordshire, UK); the Intense PulsedNeutron Source (IPNS) at Argonne National Laboratory (Argonne, Ill.),the Los Alamos Neutron Science Center (LANSCE) at Los Alamos NationalLaboratory (Los Alamos, N. Mex.), and the Neutron Science Laboratory(KENS) at KEK (Tsukuba, Ibaraki, Japan).

For a steady-state neutron source, four-circle diffractometer techniquesare used with a monochromatic beam and a single detector, rotating thecrystal and detector to measure each reflection sequentially.Diffractometer control software and step-scanning methods for intensityextraction can be adopted from routine four-circle X-ray diffractometrymethods. One or more area detectors, including area detector arrays, mayalternatively be used to increase the region of reciprocal spaceaccessed in a single measurement. A broad band (white) beam used with anarea detector allows for Laue or quasi-Laue diffraction with astationary crystal and detector.

For a pulse source with a white neutron beam, time-of-flight Lauediffraction techniques are used, which allow for the determination ofthe velocity, energy, and wavelength of each neutron detected. Thisapproach combines wavelength sorting with large area position-sensitivedetectors, and allows for fixed scattering geometries (i.e., astationary crystal and detector). Pulse source data collected in thisfashion allows for rapid collection of data sets and good accuracy andprecision in standard structural refinements. Additional detailsregarding steady-state and pulse source neutron diffraction experimentsare well known in the art. See, e.g., Chick C. Wilson, Neutron SingleCrystal Diffraction, 220 Z. Kristallogr. 385-98 (2005) (incorporated byreference herein in its entirety).

Crystal structure data, including particular isotopic ratios, areobtained from neutron diffraction data following routine structuresolution and refinement processes. Structure solution is carried outusing one of several methods, including direct methods and Pattersonmethods. For convenience, atomic coordinates from prior single crystalX-ray diffraction experiments may be used as a starting point forstructure refinement using neutron diffraction data; this approachpermits additional refinement of atomic positions, including hydrogenand deuterium positions. Refinement is conducted using full-matrixleast-squares methods to achieve optimal agreement between the observeddiffraction intensities and those calculated from the structural model.Ideally, full anisotropic refinement is carried out on all atoms,including the H/D atomic positions of interest. Data collection,structure solution and structure refinement methods, both for X-ray andneutron diffraction data, are well known in the art. See, e.g., Chick C.Wilson, Single Crystal Neutron Diffraction from Molecular Materials(World Scientific Publishing Co. 2000); George H. Stout & Lyle H.Jensen, X-Ray Structure Determination: A Practical Guide (John Wiley &Sons, Inc. 2nd ed. 1989) (both of which are incorporated herein in theirentireties).

The isotopic ratio for a particular position on a deuterated Compound Ais calculated by examining the neutron scattering cross sections for theH/D atomic position of interest. The scattering cross section isobtained as part of the refinement process discussed above. An exampleof determining the isotopic ratio for a partially deuterated compound isprovided by G. A. Jeffrey et al., Neutron Diffraction Refinement ofPartially Deuterated β-D-Arabinopyranose and α-L-Xylopyranose at 123 K,B36 Acta Crystallographica 373-77 (1980) (incorporated by referenceherein in its entirety). Jeffrey et al. used single-crystal neutrondiffraction to determine the percentage deuterium substitution forhydroxyl groups on two sugar compounds of interest. Employing themethods discussed by Jeffrey et al., one may similarly ascertain theisotopic ratio for a particular H/D position on a deuterated Compound A.

All of the cited references are incorporated herein by reference intheir entirety.

What is claimed is:
 1. A compound, wherein the compound is a compoundhaving the following structure:

or a pharmaceutically acceptable salt, stereoisomer or solvate thereof,wherein one or more of Y¹, Y², Y³, Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹,Y¹², Y¹³, Y¹⁴, Y¹⁵, Y¹⁶, Y¹⁷, Y¹⁸, Y¹⁹, Y²⁰, Y²¹, and Y²² is a hydrogenthat is isotopically enriched with deuterium, and the others of Y¹, Y²,Y³, Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵, Y¹⁶, Y¹⁷, Y¹⁸,Y¹⁹, Y²⁰, Y²¹, and Y²² are non-enriched hydrogen atoms.
 2. The compoundof claim 1, wherein the compound has the following structure:


3. The compound of claim 1, wherein one of Y¹, Y², Y³, Y⁴, Y⁵, Y⁶, Y⁷,Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵, Y¹⁶, Y¹⁷, Y¹⁸, Y¹⁹, Y²⁰, Y²¹, andY²² is isotopically enriched with deuterium, and the others arenon-enriched hydrogens.
 4. The compound of claim 1, wherein two of Y¹,Y², Y³, Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³, Y¹⁴, Y¹⁵, Y¹⁶, Y¹⁷,Y¹⁸, Y¹⁹, Y²⁰, Y²¹, and Y²² are isotopically enriched with deuterium,and the others are non-enriched hydrogens.
 5. The compound of claim 1,wherein the compound is:


6. A pharmaceutical composition comprising a compound of claim 1, or apharmaceutically acceptable salt or solvate thereof.
 7. A method oftreating a liver fibrotic disorder, diabetes or a metabolic syndromeleading to a liver fibrotic disorder comprising administering to apatient having a liver fibrotic disorder, diabetes or a metabolicsyndrome leading to a liver fibrotic disorder a compound of claim 1, ora pharmaceutically acceptable salt or solvate thereof.